Dimerization and docking domain of Bcy1, the regulatory subunit of protein kinase A from Saccharomyces cerevisiae.

ENZO TOFOLON; NICOLAS GONZALEZ BARDECI; SILVIA ROSSI; SILVIA MORENO

Rosario, Sant Fe

Simposio; 50 Reunion Nacional de SAIB; 2014

SAIB

Proteinkinases A are formed by a dimer of regulatory subunit (R2), whichbinds cAMP, bound to two catalytic subunits (C ). In mammals the N-terminus of R2is responsible for dimerization and for binding of PKA to AKAPs. Ourmodel is the R (Bcy1) subunit of PKA from S.cerevisiae. Previosuly, we have reported that: a) a mutant of Bcy1 lackingthe first 85 aa, no longer binds to specific interactors; b) a bacterialrecombinant construct of Bcy1 (1-50) contains the dimerization domain (DD)which has been structurally characterized. Using the following techniques : invitro crosslinking with EGS,  sizeexclusion chromatography, sucrose gradient sedimentation, and static lightscattering, we demonstrate that the predominant form of the recombinant DD insolution is tetrameric; this result is in accordance with the packing of the DDin the crystal structure. We have expressed a construct of the DD in a yeastvector fused to a tag of thioredoxin, HIS tag, and enterokinase cleavage site,studied the overexpression conditions, as well as its purification. We comparedthe protein profiles of Ni-agarose bound WT vs WT+DD overexpressing extractsand demonstrate that DD binds a differential set of proteins, which arecompeted by incubation with a purified preparation of whole Bcy1. These resultsindicate that in Bcy1 (1-50) resides not only the dimeration domain but aninteracting domain.